Pulmonary hypertension (PHT) is characterized by active vasoconstriction and structural changes in the small pulmonary arteries (PAs);proliferation of pulmonary artery smooth muscle cells (PASMC) contribute to the remodeling. The abnormal pathophysiology in the pulmonary vasculature may relate to decreased cyclic nucleotide levels. Phosphodiesterases (PDEs) catalyze the hydrolysis of cAMP and cGMP. In PASMC isolated from patients with PHT an increase in the expression of PDE1A and PDE1C, at least in part, accounts for lower agonist-induced cAMP levels and increased proliferation. The goal of this proposal is to study the expression, function, regulation, and therapeutic potential of PDE1 isoforms using PASMC isolated from PHT patients, and from an animal model for PHT [monocrotaline (MCT)-treated rats]. The objective of the proposal is to show that PDE1 contributes to the remodeling of the PA and that PDE1 isoforms represent novel targets for the treatment of PHT. The Specific Aims are: 1) Define the expression, localization and activity of PDE1 isoforms in human and rat PAs, focusing on PASMC, and determine if this is altered in PHT, 2) Determine the functional impact of increased PDE1 isoforms in human and rat PHT-PASMC, 3) Identify mechanisms that regulate PDE1 expression in human- and rat-PASMC, 4) Assess, using an animal model of PHT, if administration of PDE1 inhibitors can provide a therapeutic approach for PHT. Immunohistochemistry, real-time PCR, Western blotting, and PDE assays will be used to define the expression of PDE1 isoforms in human and rat lung, in particular in PASMC, and changes with PHT. The functional response of inhibition or over expression of PDE1 isoforms will be assessed by measuring cAMP, cGMP, proliferation, and apoptosis in the presence and absence of PDE1A- or PDE1 C-targeted siRNA or PDE1C adenovirus. Gel shift assay will be used to determine the transcriptional regulation of PDE1C by Nuclear factor of activated T-cells (NFAT). Finally, PDE1 inhibitors for PHT will be tested in vivo in MCT-treated rats. This proposal has the potential to reveal new aspects regarding the cellular signaling that contribute to PHT and provide novel information to support the therapeutic potential of PDE1 inhibitors in the disease, which currently has no cure.